Osteoarthritis (OA) is the most common joint disease and the leading cause of disability among Americans. OA afflicts 20 million Americans and costs $128 billion in direct medical and work-related losses each year. Nearly 1/3 of OA patients in the United States are over 65 years of age and given the aging population of the "baby boomer" generation, the prevalence of this disease is predicted to increase dramatically in the coming decades. The disease is characterized by the degeneration of cartilage and progressive loss of normal structure and function. However, the harsh loading environment and the avascular nature of mature cartilage lead to a poor intrinsic healing capacity after injury. As a result, cell-based therapies, including tissue engineering strategies for growing clinically relevant grafts, are being intensively researched. An autologous cell source would be ideal for growing clinically relevant engineered cartilage; however, using cells from an osteoarthritic or injured tissue to grow engineered cartilage with mechanical and biochemical properties similar to healthy native tissue poses several challenges, including lack of healthy donor tissues and donor site morbidity. As a result, the clinical potential of mesenchymal stem cells (MSCs) has driven forward efforts toward their optimization for tissue engineering applications. Of these MSCs, synovium-derived stem cells (SDSCs) are being intensively researched due to their proximity to the defect site and high chondrogenic potential. To address the need for cell-based therapies, functional tissue engineering aims to restore cartilage function by culturing grafts in vitro that recapitulate the mechanical, biochemical, and structural framework of the tissue in order to have an increased chance of integration and survival upon in vivo implantation. While previous work in the lab has explored the utility of physiologically relevant stimuli for creating tissue grafts with chondrocytes, it has not yet been investigated for SDSCs. Therefore, in order to determine the potential of SDSCs as a tissue engineering strategy for growing clinically relevant cartilage grafts, this dissertation had four primary aims: (1) to initially produce tissue growth utilizing synovium-derived stem cells, (2) to utilize additional chemical, physical, and physico-chemical factors to further optimize growth of tissue engineered cartilage using SDSCs, (3) to characterize the response of SDSCs to the factors applied, and (4) to utilize the optimized culture techniques to translate the findings to clinically-relevant human cells. Our initial studies investigated the potential of using physiologically relevant growth factors during both 2D expansion and 3D culture conditions, from which a baseline culture protocol was established. We then sought to explore additional strategies to further optimize tissue growth. Motivated by the discrepancy in osmolarities between native and in vitro culture conditions, we first assessed the influence of adjusting the osmolarity of the baseline culture media. We found that culturing constructs under a more physiologic osmolarity (400 mOsM) was beneficial for tissue growth. Based on these findings implicating osmolarity as a key influencer of growth potential, we sought to determine and potentially manipulate some of the pathways involved in the osmotic response in an effort to further optimize and characterize our tissue-engineered cartilage constructs. Our results supported the role of the TRPV4 ion channel in our SDSC-seeded constructs as a key mechano-osmosensing mechanism. Through the culturing techniques evaluated, we were able to achieve native mechanical and biochemical measures of juvenile bovine cartilage using SDSCs. As has been shown in the literature, observed results in other species (bovine or canine) may not always correlate to findings using human cell sources, thereby prompting the emphasis for more relevant pre-clinical models. Therefore, our final studies sought to translate our treatment strategies to clinically relevant human cells from normal (non-diseased) and diseased (OA) SDSCs and chondrocytes in order to determine their utility. We were able to create a complete set of micropellet data for both SDSCs and chondrocytes to allow for comparisons. Overall, our micropellet results indicate that tissue condition (non-diseased vs OA) is the primary determinant of matrix synthesis. The research described in this dissertation has demonstrated the utility of SDSCs for strategies aimed at cartilage regeneration. We present the first studies to grow SDSC-seeded constructs to native properties of juvenile bovine chondrocytes. Therefore, utilization of the culture techniques presented here and other optimization strategies may hold key insights to developing a tissue using autologous/allogeneic SDSCs that can fully recreate native cartilage. In addition, the findings support the clinical potential of human SDSCs as a cell source for cartilage repair strategies.

The overall goal of this thesis is to develop a new non-contact, whole-body, fluorescence molecular tomography system for small animal imaging. Over the past decade, small animal in vivo imaging has led to a better understanding of many human diseases and improved our ability to develop and test new drugs and medical compounds. Among various imaging modalities, optical imaging techniques have emerged as important tools. In particular, fluorescence and bioluminescence imaging systems have opened new ways for visualizing many molecular pathways inside living animals including gene expression and protein functions. While substantial progress has been made in available prototype and commercial optical imaging systems, there still exist areas for further improvement in the outcome of existing instrumentations. Currently, most small animal optical imaging systems rely on 2D planar imaging that provides limited ability to accurately locate lesions deep inside an animal. Furthermore, most existing tomographic imaging systems use a diffusion model of light propagation, which is of limited accuracy. While more accurate models using the equation of radiative transfer have become available, they have not been widely applied to small animal imaging yet. To overcome the limitations of existing optical small animal imaging systems, a novel imaging system that makes use of the latest hardware and software advances in the field was developed. At the heart of the system is a new double-conical-mirror-based imaging head that enables a single fixed position camera to capture multi-directional views simultaneously. Therefore, the imaging head provides 360-degree measurement data from an entire animal surface in one step. Another benefit provided by this design is the substantial reduction of multiple back-reflections between the animal and mirror surfaces. These back reflections are common in existing mirror-based imaging heads and tend to degrade the quality of raw measurement data. Furthermore, the conical-mirror design offers the capability to measure angular-resolved data from the animal surface. To make full use of this capability, a novel equation of radiative transfer-based ray-transfer operator was introduced to map the spatial and angular information of emitted light on the animal surface to the captured image data. As a result, more data points are involved into the image reconstructions, which leads to a higher image resolution. The performance of the imaging system was evaluated through numerical simulations, experiments using a well-defined tissue phantom, and live-animal studies. Finally, the double reflection mirror scheme presented in this dissertation can be cost-effectively employed with all camera-based imaging systems. The shapes and sizes of mirrors can be varied to accommodate imaging of other objects such as larger animals or human body parts, such as the breast, head, or feet.

The rate limiting factor for the treatment of neurodegenerative diseases is the blood-brain barrier (BBB), which protects the brain microenvironment from the efflux of large molecules, and thus it constitutes a major obstacle in therapeutic drug delivery. All state-of-the-art strategies to circumvent the BBB are invasive or non-localized, include side-effects and limited distribution of the molecule of interest to the brain. Focused Ultrasound (FUS) in conjunction with microbubbles has been shown to open the BBB non-invasively, locally and transiently to allow large molecules diffusion in rodents and non-human primates. This thesis entails a quantitative analysis of the FUS-induced BBB opening in vivo for drug delivery in neurodegenerative diseases. First, quantitative analysis and modeling of the physiologic changes of the BBB opening, such as permeability changes, volume of opening, and reversibility timeline, were studied in wild-type mice, in brain areas related to Alzheimer's and Parkinson's disease. This study provided in vivo tools for BBB opening analysis, as well as the design of a FUS method with optimized parameters for efficient and safe drug delivery. Second, the neurotrophic factor Neurturin, which has been shown to have neuroregenerative and neuroprotective effects in dopaminergic neurons was successfully delivered in wild-type mice and MPTP-lesion parkinsonism model mice. It was shown that FUS enhanced the delivery of Neurturin to the entire regions of interest associated with the disease, downstream signaling for neuronal proliferation was also detected, and finally neuroregeneration was observed in the FUS-treated side compared to the contralateral side. In the third part of this thesis, a pre-clinical translation of the pharmacodynamic analysis was designed and analyzed in non-human primates. The permeability changes, the volume of opening separately in grey and white matter, as well as the concentration of an MR-contrast agent were measured in vivo for the first time. The interaction of FUS with the inhomogeneous primate brain was investigated and the drug delivery efficiency of the FUS technique for BBB opening was measured non-invasively; rather critical findings for safe and optimal drug delivery using FUS in a pre-clinical setting.

Preclinical screening during the development of new drugs is poorly predictive and costly, creating a significant interest from pharmaceutical companies, government agencies, and the public in the development of better preclinical tests. To create more predictive organ models, human derived stem cells can be coupled with biomimetic tissue engineering approaches to create physiologically relevant functional subunits of each tissue/organ within the body. However, existing methods of generating cardiomyocytes (CMs) and cardiac tissues from human induced pluripotent stem cells (hiPSC) derived CMs (hiPS-CMs) are relatively immature and produce tissues that resemble that of a fetal heart at best. This limits their use in therapeutic development and thus, methods to overcome their immature phenotype are of high importance. In pursuit of this goal, this dissertation focuses on the role of biophysical stimuli in driving the functional maturation of hiPSC-CMs to engineer cardiac muscle of high biological fidelity. In an effort to recapitulate the hierarchical structure and functionality of native heart tissue, methods to pattern cells at the nano- and microscale levels were developed and optimized towards the functional assembly of cardiac tissues at the macroscale. To address the challenges currently associated with hiPS-CM immaturity, the decoupled effects of electrical and electromechanical stimulation in driving cardiac maturation were investigated. Subsequently, optimal electromechanical stimulation regimens were established. Daily intervals of high intensity electromechanical training were shown to upregulate cardiac functionality and energetics, and thus, enhance maturation. Combining these methods enabled the development of a custom bioreactor capable of generating larger, more functionally mature hiPS-CM tissues. Mimicking the developmental increases in cardiac beating frequency, exposure of the resulting tissues to a dynamic electromechanical intensity training regimen matured hiPS-CMs beyond levels currently demonstrated within the field. Specifically, the engineered tissues recapitulated many of the molecular, structural, and functional properties of adult human heart muscle, including well developed registers of sarcomeres, networks of T-tubules, calcium homeostasis, and a positive force-frequency relationship. The enhanced functionality of the resulting bio-engineered adult-like myocardium enabled its utility in predicting drug cardiotoxicity and modeling human cardiac disease.

Cartilage is a tissue that is critical for skeletal function, yet its study has been limited by a lack of quantitative, non-destructive, three-dimensional imaging techniques that enable simultaneous interrogation of both bone and cartilage. Recently, methods of contrast-enhanced micro-computed tomography (CECT) have been developed that exploit the electrostatic interactions between ionic contrast agents and negatively charged glycosaminoglycans (GAGs) in cartilage, thus providing information about the composition and morphology of cartilage that was previously only available via destructive methods. The goal of this dissertation project was to apply CECT, a non-destructive, three-dimensional imaging method, to understand the how the morphology and composition of cartilage changes in response to injury and disease. First, CECT was applied to a model of growth plate injury to quantify changes to the cartilaginous tissue of the growth plate and formation of bone bridges within this tissue in response to injury. Using CECT, it was possible to identify increased thickness and CECT attenuation at the injury site. This result, paired with histological evidence of localized dysregulation of cellular activity, suggests that treatment designed to reduce bone bridge formation at the injury site should also consider the effects of the treatment on the adjacent cartilage. Second, CECT was applied to a collagen antibody-induced arthritis (CAIA) model to determine the role of the A2B adenosine receptor (A2BAR) in the arthritic deterioration of bone and cartilage. CECT scans demonstrated that loss of GAG in the cartilage preceded degeneration, but that ablation of the A2BAR in mice had little effect on the degenerative changes in bone and cartilage associated with CAIA. These results suggest that the A2BAR does not independently mediate these changes and that it may be necessary to target multiple adenosine receptors. Third, the ability of CECT to monitor the fracture healing and predict the stiffness of the cartilaginous fracture callus was assessed both at the level of the whole callus and at the level of the cartilage tissue. Callus stiffness was negatively correlated with the size of the callus and the amount of cartilage, while neither stiffness nor indentation modulus were correlated with CECT attenuation, suggesting that the stiffness of the cartilaginous fracture callus depends on the amount, rather than GAG content of cartilage. The work presented in this dissertation provides outlines changes in both bone and cartilage that occur in pathological conditions and provides new insights for both the treatment and assessment of these conditions.

Cancer accounts for the highest amounts of disease related premature deaths worldwide with 8.1 million of deaths being associated with malignancy. In the cancer microenvironment, particularly in solid tumours, hypoxia plays a significant role in progression and metastasis by altering signal transduction and gene regulation which leads to aggressive phenotypes, poor prognosis and lower survival rates. Hypoxia-Inducible Factor (HIF) driven gene regulation is well established and believed to promote survival of tumour cells under the hypoxic microenvironment. Accumulating evidence also suggests that Specificity protein (Sp) family transcription factors might also play a role in the hypoxic microenvironment by regulating transcription of key hypoxia responsive genes such as VEGFA in either a HIF dependent or independent manner. In normal cells, Sp transcription factors are ubiquitously expressed and known to regulate numerous genes involved in vital cellular pathways such as cell cycle, apoptosis and angiogenesis. In tumour environments, deregulated Sp protein levels have been demonstrated and shown to correlate with poor prognosis and treatment response. However, the exact role of Sp transcription factors in hypoxic microenvironment is not fully understood. This study aimed to identify the effect of severe (chronic) hypoxia on Sp transcription factors and hypoxia-responsive gene regulation using breast cancer as a cell model. Initial studies measured the expression levels and binding activity of Sp transcription factors. Subsequently, an integrative genomic analysis was performed to identify Sp driven hypoxia-responsive genes in breast cancer cells. The study was further extended to analyse the binding kinetics of Sp protein inhibitors using surface plasmon resonance spectroscopy. Finally, transcriptional changes of hypoxia responsive genes were examined after addition of Sp inhibitors or knockdown of Sp1 level under the hypoxic environment. Expression analysis of Sp family members (Sp1-4) showed that the transcript levels of Sp genes were unaffected due to chronic hypoxic exposure whilst Sp protein levels were induced in all three cell lines. However, expression patterns were dependent on tissue type and severity of hypoxia. Twenty genes were identified as potential Sp driven hypoxia responsive genes which are consist of GC-rich putative Sp binding sites in their promoters. Gene expression analysis validated the hypoxic induction of these genes and their dependency on Sp protein-mediated transcription. Affinity studies of Sp protein inhibitors prove binding of antibiotic derivatives, Mithramycin A and Chromomycin A to GC-rich regions with different binding affinities and kinetics. Different Equilibrium constants (Kd) of Mithramycin A and Chromomycin A were identified which varied according to the promoter sites (10−3 to 10−6 M range). Furthermore, novel data also confirm the Mithramycin-DNA interaction is independent of cation Mg2+ which has been considered obligatory for DNA interaction. Interestingly, nordihydroguaiaretic acid (NDGA) derivative, Terameprocol exhibits no detectable interaction with linear DNA consisting of Sp binding sites. These results emphasise the importance of Sp proteins as regulators of hypoxia-mediated gene transcription. Sp-regulated transcription is vital in altering hypoxia-related cellular pathways and has a potential as biomarkers for solid tumours. Moreover, these results suggest the potential use of Sp antagonists to inhibit expression of key hypoxic genes in the cancer microenvironment. Results also provide solid background knowledge on pharmacokinetics of Sp inhibitors which will be useful in synthesis of new derivatives which can be used in novel therapeutic strategies for cancer and perhaps other diseases. Understanding the molecular mechanisms of Sp mediated hypoxic gene regulation can be further extended to elucidate other cellular stress and cellular adaptive mechanism.

Biomedical imaging is a key tool for the study of host-pathogen interactions. New techniques are enhancing the quality and flexibility of imaging systems, particularly as a result of developments in laser technologies. This work applies the combination of two advanced laser imaging methods to study the interactions between a virus and the host cells it infects. The first part of this work describes the theory and experimental implementation of coherent anti-Stokes Raman scattering microscopy. This technique-first demonstrated in its current form in 1999-permits the imaging of microscopic samples without the need for fluorescent labelling. Chemical contrast in images arises from the excitation of specific vibrations in the sample molecules themselves. A laser scanning microscope system was set up, based on an excitation source consisting of two titanium-sapphire lasers synchronized with a commercial phase-locked loop system. A custom-built microscope was constructed to provide optimal imaging performance, high detection sensitivity and straightforward adaptation to the specific requirements of biomedical experiments. The system was fully characterized to determine its performance. The second part of this work demonstrates the application of this microscope platform in virology. The microscope was configured to combine two nonlinear imaging modalities: coherent anti-Stokes Raman scattering and two-photon excitation. Mouse fibroblast cells were infected with a genetically modified cytomegalovirus. The modification causes the host cell to express the green fluorescent protein upon infection. The host cell morphology and lipid droplet distribution were recorded by imaging with coherent anti-Stokes Raman scattering, whilst the infection was monitored by imaging the viral protein expression with two-photon excitation. The cytopathic effects typical of cytomegalovirus infection were observed, including expansion of the nucleus, rounding of the cell shape, and the appearance of intracellular viral inclusions. In some cases these effects were accompanied by dense accumulations of lipid droplets at the nuclear periphery. Imaging was performed both with fixed cells and living. It was demonstrated that the lipid droplets in a single live cell could be imaged over a period of 7 hours without causing noticeable laser-induced damage. The system is shown to be a flexible and powerful tool for the investigation of virus replication and its effects on the host cell.

Nanoparticle (NP) drug delivery is a major focus in the research community because of its potential to use existing drugs in safer and more effective ways. Chemotherapy encapsulation in NPs shields the drug from the rest of the body while it is within the NP, with less systemic exposure leading to fewer off-target effects of the drug. However, passive loading of drugs into NPs is a suboptimal method, often leading to burst release upon administration. This work explores the impact of incorporating the drug-polymer conjugate doxorubicin-poly (lactic-co-glycolic) acid (Dox-PLGA) into a lipid-polymer hybrid nanoparticle (LPN). The primary difference in using a drug-polymer conjugate for NP drug delivery is the drug’s release kinetics. Dox-PLGA LPNs showed a more sustained and prolonged release profile over 28 days compared to LPNs with passively loaded, unconjugated doxorubicin. This sustained release translates to cytotoxicity; when systemic circulation was simulated using dialysis, Dox-PLGA LPNs retained their cytotoxicity at a higher level than the passively loaded LPNs. The in vivo implication of preserving cytotoxic potency through a slower release profile is that the majority of Dox delivered via Dox-PLGA LPNs will be kept within the LPN until it reaches the tumor. This will result in fewer systemic side effects and more effective treatments given the higher drug concentration at the tumor site. An intriguing clinical application of this drug delivery approach lies in using Dox- PLGA LPNs to cross the blood-brain barrier (BBB). The incorporation of Dox-PLGA is hypothesized to have a protective effect on the BBB as its slow release profile will prevent drug from harming the BBB. Using induced pluripotent stem cells differentiated to human brain microvascular endothelial cells that comprise the BBB, the Dox-PLGA LPNs were shown to be less destructive to the BBB than their passively loaded counterparts. Dox-PLGA LPNs showed superior cytotoxicity against plated tumor cells than the passively loaded Dox LPNs after passing through an in vitro transwell BBB model. Dox-PLGA LPNs and drug-polymer conjugates are exciting alternatives to passively loaded NPs and show strong clinical promise of a treatment that is more potent with fewer side effects and less frequent administration.

Computational methods for predicting macromolecular complexes are useful tools for studying biological systems. They are used in areas such as drug design and for studying protein-protein interactions. While considerable progress has been made in this field over the decades, enhancing the speed and accuracy of these computational methods remains an important challenge. This work describes two different enhancements to the accuracy of ClusPro, a method for performing protein-protein docking, as well as an enhancement to the efficiency of global rigid body docking. SAXS is a high throughput technique collected for molecules in solution, and the data provides information about the shape and size of molecules. ClusPro was enhanced with the ability to SAXS data collected for protein complexes to guide docking by selecting conformations by how well they match the experimental data, which improved docking accuracy when such data is available. Various other experimental techniques, such as NMR, FRET, or chemical cross linking can provide information about protein-protein interfaces, and such information can be used to generate distance-based restraints between pairs of residues across the interface. A second enhancement to ClusPro enables the use of such distance restraints to improve docking accuracy. Finally, an enhancement to the efficiency of FFT based global docking programs was developed. This enhancement allows for the efficient search of multiple sidechain conformations, and this improved program was applied to the flexible computational solvent mapping program FTFlex. / 2018-07-09T00:00:00Z

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